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Homing CRISPR/Cas9 Technology to Barcode and Lineage Trace Mouse Development



Review of “Developmental barcoding of whole mouse via homing CRISPR” from Science by Stuart P. Atkinson 

The recent development of technology allowing in vivo barcode generation has provided researchers with the potential ability to track and visualize every cell division from a single totipotent zygote to a fully-fledged organism [1-3]. However, while studies have reported recording and lineage tracing in cultured cells and lower vertebrates, the application of this amazing technology has yet to reach an animal model with relevance to human health.

Researchers from the laboratory of George M. Church (Harvard University, Boston, MA, USA) recently rose to meet this vexing challenge, and now report in Science on the reconstruction of early lineages and the investigation of axis development in the brain following robust in vivo barcoding and lineage recording in mice by homing CRISPR/Cas9 technology [4].

The in vivo barcoding strategy described employed a mouse line carrying multiple genomically integrated homing CRISPR guide RNA (hgRNA) loci, generated via the transfection of an hgRNA transposon library into mouse embryonic stem cells and their subsequent injection into blastocysts to create chimeric mice. hgRNAs, modified single guide RNAs (sgRNAs), target their own loci to establish a broad diversity of mutants that combine to form genetic barcodes [5]. The crossing of this strain with a Cas9-expressing mouse strain kick-starts continuous mutagenic activity after conception and creates developmentally barcoded mice with information recorded in lineage-specific mutations. This system generates unique accumulative mutation profiles in each lineage with closely related cells displaying a more similar mutation profile than distantly related cells.

Excitingly, the authors employed this new strategy to construct a robust lineage tree of the early stages of development; that is, the differentiation of the blastomere into the trophectoderm and inner cell mass (ICM) and the subsequent differentiation of the ICM into the primitive endoderm and epiblast. Furthermore, assessments of subsequent developmental stages also established that commitment to the anterior-posterior axes in the brain occurs before commitment to the lateral axes during the development of the central nervous system.

The authors note that homing CRISPR/Cas9 strategy for in vivo barcoding and lineage tracing in a mammalian model system may aid the understanding of disease-causing developmental aberrations, the restoration of function to damaged/diseased tissues, and the generation of tissues and organs from stem cells. Moreover, they also highlight other potential applications in the future, including the recording of cellular signals over time and uniquely barcoding each cell in a tissue/organism for identification purposes.

For more on all the new CRISPR/Cas9-based techniques and further applications of in vivo barcode generation, stay tuned to the Stem Cells Portal.


  1. Peikon ID, Gizatullina DI, and Zador AM, In vivo generation of DNA sequence diversity for cellular barcoding. Nucleic Acids Research 2014;42:e127-e127.
  2. Ma J, Shen Z, Yu Y-C, et al., Neural lineage tracing in the mammalian brain. Current Opinion in Neurobiology 2018;50:7-16.
  3. Woodworth MB, Girskis KM, and Walsh CA, Building a lineage from single cells: genetic techniques for cell lineage tracking. Nature Reviews Genetics 2017;18:230.
  4. Kalhor R, Kalhor K, Mejia L, et al., Developmental barcoding of whole mouse via homing CRISPR. Science 2018;361.
  5. McKenna A, Findlay GM, Gagnon JA, et al., Whole-organism lineage tracing by combinatorial and cumulative genome editing. Science 2016;353:aaf7907.